U.S. patent number 6,020,305 [Application Number 08/689,277] was granted by the patent office on 2000-02-01 for treatment of stress-induced skin disease by corticotropin releasing hormone antagonists and skin mast cell degranulation inhibitors.
This patent grant is currently assigned to Kos Pharmaceuticals, Inc.. Invention is credited to Theoharis C. Theoharides.
United States Patent |
6,020,305 |
Theoharides |
February 1, 2000 |
Treatment of stress-induced skin disease by corticotropin releasing
hormone antagonists and skin mast cell degranulation inhibitors
Abstract
A method of reducing or blocking a stress-related atopic skin
disease such as exzema or uticaria in a subject comprising
administering to the patient an agent that antagonizes CRH-induced
activation of skin mast cells, the agent being used alone or
together with a second agent that inhibits activation of skin mast
cells. Such agents include compositions that reduce the production
or secretion of CRH, neurotensin or somatostatin or an agent that
inhibitos the physiological action of CRH, neurotensin or
somostatin on skin mast cells. The effects of CRH on skin mast
cells can also be inhibited by histamine-3 receptor antagonists and
by inhibitors of the phosphorylation of skin mast cell moesin.
Inventors: |
Theoharides; Theoharis C.
(Brookline, MA) |
Assignee: |
Kos Pharmaceuticals, Inc.
(Miami, FL)
|
Family
ID: |
24767751 |
Appl.
No.: |
08/689,277 |
Filed: |
August 6, 1996 |
Current U.S.
Class: |
424/158.1;
514/10.8; 514/11.1; 514/18.6; 514/18.7; 514/456; 514/653;
514/9.7 |
Current CPC
Class: |
A61K
38/2228 (20130101); A61K 45/06 (20130101); A61K
31/352 (20130101); A61K 31/495 (20130101); A61K
38/2228 (20130101); A61K 31/352 (20130101); A61K
31/495 (20130101); A61K 2300/00 (20130101); A61K
2300/00 (20130101); A61K 2300/00 (20130101) |
Current International
Class: |
A61K
38/22 (20060101); A61K 037/00 (); A61K
031/35 () |
Field of
Search: |
;514/2,456,653 |
Foreign Patent Documents
Other References
Shapiro et al. Pharmacotherapy 5(3): 156-170 (1985). .
Webster et al. Endocrinology 137(12): 5747-5750 (1996). .
Tauberg et al. Anesthesia Progress XXX(6): 199-200 (1983). .
Koblenzer Psychodermatology 14(3): 447-455 (1996)..
|
Primary Examiner: Fay; Zohreh
Attorney, Agent or Firm: Foley & Lardner
Claims
What is claimed is:
1. A method for reducing or blocking atopic skin disorders in a
subject, comprising the step of administering to said subject an
effective amount of an antagonist of the activation of skin mast
cells by CRH, wherein said antagonist is selected from the group
consisting of anti-CRH antibody, competitive and non-competitive
inhibitors of the binding of CRH to skin mast cell CRH receptors,
agents that reduce the production or release of CRH in the skin of
said subject, inhibitors of the action of neurotensin on skin mast
cells, and agents that reduce the production or release of
neurotensin.
2. The method of claim 1, wherein said antagonist is an anti-CRH
antibody.
3. The method of claim 1, wherein said antagonist is a competitive
or non-competitive inhibitor of the binding of CRH to skin mast
cell CRH receptors.
4. The method of claim 1, wherein said antagonist reduces the
production or release of CRH in the skin of said subject.
5. The method of claim 1, wherein said antagonist is an inhibitor
of the action of neurotensin on skin mast cells.
6. The method of claim 5, wherein said antagonist reduces the
production or release of neurotensin in the skin of said
subject.
7. The method of claim 1, further comprising administering a second
inhibitor along with said CRH antagonist, wherein said second
inhibitor is either an inhibitor of mast cell degranulation or an
antagonist of a histamine receptor.
8. The method of claim 7, wherein said second inhibitor is a
histamine-3 receptor agonist.
9. The method of claim 7, wherein said said second inhibitor is a
piperazine compound.
10. The method of claim 9, wherein said piperazine compound is
hydroxyzine or an analogue thereof.
11. The method of claim 7, wherein said second inhibitor is an
agent that stimulates the phosphorylation of at least one serine or
threonine residue in skin mast cell moesin.
12. The method of claim 11, wherein said agent is a bichromone.
13. The method of claim 12, wherein said bichromone is cromolyn or
an analogue thereof.
14. The method of claim 11, wherein said agent is a flavonoid.
15. The method of claim 14, wherein said flavonoid is
kaempferol.
16. The method of claim 1, wherein said antagonist is an inhibitor
of the production of somatostatin or action of somatostatin on skin
mast cells.
17. The method of claim 1 or claim 7, wherein said antagonist or
said second inhibitor or both in a pharmaceutically acceptable
vehicle is administered to said subject by a route selected from
the group consisting of parenteral, oral, sublingual, topical and
transdermal.
Description
BACKGROUND OF THE INVENTION
The invention relates in general to treatment of atopic, stress
related skin disease. In particular, the invention relates to
effects of Corticotropin-Releasing Hormone ("CRH") on skin mast
cells that result in various skin disorders, and the use of
inhibitors and antagonists of CRH to treat such conditions.
CRH is a major regulator of the hypothalamic-pituitary-adrenal axis
and principal coordinator of the stress response. While the
neuroendocrine release of CRH results in anti-inflammatory effects
through activation of the hypothalamic-pituitary-adrenal axis and
the sympathetic nervous system, recent evidence suggests that CRH
is also secreted peripherally and may have pro-inflammatory
actions. Immunoreactive CRH (iCRH) has been localized to immune
accessory cells in induced aseptic joint inflammation, in
streptococcal arthritis, as well as expermental uveitis. CRH can be
synthesized locally at inflammatory sites as evidenced by the
presence of CRH mRNA in chronically inflamed synovia in rats or
released by post-ganglionic sympathetic neurons and sensory
afferent fibers. Immunocytochemistry has also verified the presence
of iCRH in human tissues undergoing inflammatory processes.
Both iCRH and CRH mRNA have been demonstrated in rat and mouse
spleen and thymus, as well as in human peripheral blood leukocytes,
while mitogenic stimulation of human T lymphocytes results in
synthesis of CRH. However, there is a discrepancy between the
abundance of iCRH and the paucity of its mRNA at inflammatory sites
in the early, acute phase of inflammation. The demonstration of
CRH-like immunoreactivity in the dorsal horn of the spinal cord and
dorsal root ganglia, as well as in sympathetic nerve cell bodies
and sympathetic ganglia, support the hypothesis that the majority
of iCRH in early inflammation is of nerve cell rather than immune
cell origin. For purposes of the present invention, it is important
that CRH and CRH-receptor mRNA have been identified in human skin
(Slominski et al., FEBS Letts., 374:113 (1995)).
CRH administration to humans or animals causes significant
peripheral vasodilation manifested as increased blood flow and
flushing. Immune CRH also appears to have pro-inflammatory actions
as systemic administration of rabbit anti-CRH serum suppressed both
the amount of exudate and inflammatory cell accumulation in induced
inflammation (Karalis et al., Science, 254: 421(1991)), as well as
the severity of experimentally-induced uveitis. Moreover, when this
antiserum was administered prior to acute psychological stress, it
blocked the resultant activation of dura mast cells (Theoharides et
al., Endocrinol. 136:5745 (1995). Analogous to the marked
appearance of iCRH, there is activation and proliferation of mast
cells at inflammatory sites.
Mast cells are located in the perivascular area close to peripheral
nerves and, when activated by nerve stimulation or sensory
neuropeptides, they secrete potent vasoactive and proinflammatory
mediators including histamine, cytokines, prostanoids and
proteases. In fact, tumor necrosis factor (TNF) released from skin
mast cells induces both vasodilation and expression of endothelial
adhesion molecule-1, while skin mast cell activation by substance P
(SP) leads to granulocyte infiltration.
In the light of this background, it is an object of this invention
to discover whether iCRH may be involved in the pathophysiology of
certain skin conditions that are exacerbated by stress, such as
eczema and urticaria. CRH could interact with the immune and the
nervous systems and contribute to inflammation, possibly through
activation of mast cells. Such a functional relation between
central CRH, iCRH, neurons and mast cells could be important in
normal physiology, in the context of hypersensitivity reactions,
and in neuroimmunoendocrine or inflammatory syndromes. A further
object of this invention was to test this hypothesis by examining
the ability of CRH and stress to activate skin mast cells. It is an
additional object of this invention to provide treatments that
block or inhibit the effects of CRH on mast cell related skin
disorders.
SUMMARY OF THE INVENTION
These objects have been achieved by the discovery that CRH causes
activation of skin mast cells and skin vasodilation, an effect that
is reproduced by acute psychological stress due to immobilization.
The invention comprises treatment of stress-exacerbated skin
disorders in a subject by the administration of substances that
antagonize the physiological effects of CRH on skin mast cells.
In one embodiment of the invention, anti-CRH antibodies are
administered to a subject to treat CRH-related skin disorders.
In another embodiment, blocking anti-CRH receptor antibodies or
anti-receptor antagonists are administered to a subject to treat
CRH-related skin disorders.
In still another embodiment, antagonists of neurotensin ("NT") and
NT receptors are administered to block the potentiating effect of
NT on the action of CRH on skin mast cells.
In yet another embodiment, pharmaceutical inhibitors of skin mast
cell degranulation are administered either alone or with the CRH
and NT antagonists mentioned above.
In a yet further embodiment, combinations of CRH antagonists and
skin mast cell degranulation inhibitors are used to treat atopic
skin disorders.
These and other embodiments will become apparent by reference to
the specification and appended claims.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
This invention is based on the present discovery that
stress-related skin disorders such as eczema and uticaria in a
human subject can be treated by administering to the subject an
antagonist of the physiological actions of CRH on skin mast cells.
The term "antagonist" is used herein to include agents that inhibit
or block the synthesis or release of CRH from tissue sources or
that reduce the physiological effects of CRH as an activator of
skin mast cell degranulation.
The aforementioned CRH antagonists include compositions that
directly or indirectly reduce CRH release from neurons or other
sources or inhibit CRH actions on skin mast cells. Such antagonists
include an anti-CRH antibody or a structural analogue of CRH, which
includes amino acid substitutions and derivatization. The
antagonists may be agents that block the activity of CRH on skin
mast cell receptors, such as anti-CRH receptor antibodies. The
antagonists may be compounds that block or antagonize the actions
of biochemicals that mediate or promote CRH actions, such as an
anti-neurotensin ("NT") antibody or an NT receptor antagonist. The
antagonist may be an agent that inhibits skin mast cell activation,
such as a bichromone (e.g. cromolyn (disodium cromoglycate)), a
flavonoid (kaempferol), other compounds that stimulate the
phosphorylation of the 78 kDa mast cell moesin (see pending U.S.
Ser. No. 08/631,184), a piperazine (e.g., hydroxyzine), or a
histamine-3 receptor agonist (N.sup..mu. -methyl histamine,
Calbiochem/Novabiochem, La Jolla, Calif.).
As noted above, inhibition of the effect of CRH in the development
of atopic skin disorders may be achieved by treating a subject with
an an antagonist of the production or release of CRH from neurons
and other sites of origin, by inhibiting the binding of CRH to mast
cell CRH receptors, and by inhibiting the physiological action(s)
of CRH on mast cells (for example, by an antiserum to NT or an
anti-NT receptor antibody administered parenterally or topically).
Although applicant need not be bound by any particular theory of
mechanism of action, it is likely that the anti-CRH antibody by
binding to CRH, and an anti-CRH receptor antibody or CRH receptor
blocker by blocking the binding of CRH to its physiological
receptor(s) on skin mast cells, thereby inhibits or blocks CRH
activation of skin mast cells.
Other CRH antagonists are expected to have the same beneficial
effects as the anti-CRH antibody, and therefore are within the
scope of this invention. Examples include a CRH receptor antagonist
such as the CRH peptide analogue D-Phe.sup.12, Nle.sup.32, .sup.21,
Ala.sup.38 hCRH (12-41)NH2 (Neurocrine Biochemicals, Inc., cat. no.
1P-36-41, MW 3474.1), and Pfizer's non-peptide CRH analog
CP-154,526-1 that will compete with CRH for binding to CRH
receptor(s) on mast cells. Other examples include inhibitors of CRH
secretion such as agents that activate histamine-3 receptors.
Anti-CRH polyclonal serum may be prepared by routine immunization
of rabbits with commercially available recombinant CRH (rCRH)
(Sigma Chem. Co., St. Louis, Mo.) according to Karalis et al.,
Science, 254:421 (1991), which is incorporated herein by reference
in its entirety, or obtained from Phoenix Pharmaceuticals,
California. A rabbit anti-NT receptor serum can be purchased from
Sanofi Research, Toulouse, France, which is also the source of the
nonpeptide NT receptor antagonist SR 48692.
Anti-CRH and anti-NT polyclonal or monoclonal antibodies or other
inhibitors may be administered parenterally, orally, sublingually,
topically or transdermally to subjects in a pharmaceutically
acceptable carrier such as those described in Remington's
Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1988,
which is incorporated herein by reference. For parenteral use,
concentrations of about 0.1 to 10 mg protein/ml for anti-CRH and
anti-NT sera, and at a concentration of 1 to 1000 nM for the NT
receptor antagonist SR 48692 or CRH receptor antagonists shown
above may be used. Cromolyn (Fisons, Inc.) in a pharmaceutically
acceptable vehicle may be administered to patients in two 100 mg
capsules before meals and at bedtime. Transdermal injections or
patches may also be used. Physicians of skill in this art will,
without undue experimentation, select the proper inhibitors and the
appropriate dosages and routes of administration for the particular
clinical picture.
Although skin mast cell activation can be determined histologically
by light or electron microscopy, or by dye extravasation, as
described below, it can also be determined by assaying peripheral
blood by routine methods for the appearance of the products of
activation, such as histamine, its metabolite N-methyl-histamine,
or the proteolytic enzyme tryptase as described in Roznecki et al.,
Ann. Neurol. 37:63 (1995) which is incorporated herein by reference
in its entirety.
The CRH receptor cDNA has been cloned from a variety of animal
species and organs. Perris et al., PNAS USA, 92:2968 (1995). CRH
receptors range in size from 415 to 431 amino acid residues with
68% homology between rodent and human molecules. The exact type of
skin cells expressing CRH receptors may, therefore, be determined
by in situ hybridization and reverse transcriptase PCR (Slominski
et al., FEBS Letts. 374:113 (1995)).
The examples that follow are designed to exemplify the invention
and are not intended to limit the scope of the invention, which is
described in the specification and appended claims.
The following materials and methods were employed in the examples
described below:
1. For extravasation experiments, male Sprague/Dawley rats, each
weighing approximately 350 g (Taconic Farms, Germantown, N.Y.),
were anesthetized with a single intraperitoneal injection of 0.5 ml
ketamine and 0.5 ml xylazine (20 mg/ml each) and then injected
intravenously with 0.1 ml of 1% Evan's blue 60 min prior to
treatment.
2. Drugs tested by intradermal injection in a volume of 0.1 ml
include: a) 0.1 .mu.g/ml of the mast cell specific secretagogue,
compound 48/80 (C48/80), b) 10.sup.-5 M substance P (Sigma), c)
various concentrations of CRH (Sigma, St. Louis), d) the CRH
receptor antagonist [D-Phe.sup.12 Nle.sup.21,38, Ala.sup.32
]rCRH(12-41), e) the inactive free acid form of recombinant/human
CRH (r/h CRH--OH), both obtained from Neurocrine Biosciences, CA,
f) somatostatin (somatotropin release inhibitory factor, SRIF
Bachem, Calif.), g) diphenylhydramine (Sigma), H) terfenadine
(Sigma), i) hydroxyzine (UCB, Belgium) and j) the antiserum to the
78 kDa portion of the Tumor Necrosis Factor receptor (Harland
Serolab, Ltd., Crawley Down, Sussex, England), in normal saline
using a tuberculin syringe.
3. The pretreatment solution, when appropriate, was drawn first in
the syringe followed by CRH from which it was separated by an air
bubble. The pretreatment solution was injected first and was
allowed to remain in the skin for 1 min; the needle was then
changed, the air bubble ejected and CRH was injected and allowed to
stay in the skin for 5 min. The animal was then killed by
asphyxiation over CO.sub.2 vapor and decapitated; the skin was
removed, turned over and photographed.
4. In other rats, the skin was rapidly removed at the end of the
injection period and was fixed in 4% paraformaldehyde for light
microscopy (Theoharides et al., Int. Arch. Allergy Immunol. 102:352
(1993)), which is incorporated herein by reference in its entirety.
The tissue was then frozen and thin sections (7 .mu.) were cut
using a cryostat (Jung CM 3000, Leica, Luc. Deerfield, Ill.). The
sections were stained with acidified (pH<2.5) toluidine blue
(Sigma, St. Louis) and all mast cells were counted, by two
different researchers blinded to the experimental conditions, at
400.times. magnification using a Diaphot inverted Nikon microscope
(Don Santo, Mass.).
5. For depletion of neuropeptide-containing sensory nerves, one
entire litter of rats was injected with capsaicin within two days
of birth and the males were used five weeks later as described
before (Dimitriaou et al., Neuroscience 44: 97 (1991), which is
incorporated by reference herein in its entirety. The effectiveness
of this procedure was confirmed by immunocytochemistry (Dimitraou
1991 above}, which showed that there were no Substance P-positive
cells or nerve processes in the skin of capsaicin-treated rats.
EXAMPLE 1
CRH and Skin Vascular Permebility
CRH induced marked increases in skin vascular permeability, as
evidenced by dye extravasation, an effect which was more pronounced
than that obtained by an equimolar concentration of the mast cell
secretagogue C48/80 or SP (Table 1).
EXAMPLE 2
CRH and Skin Mast Cell Activation
The CRH effect on skin mast cell activation was confirmed by
morphological evidence of secretion as judged by granule content
extrusion and loss of cellular staining in the skin samples from
the injection site. Activation was observed in about 45% of the
mast cells at skin sites treated with 10.sup.-4 M CRH (3 rats,
n=841), as compared to 28% (3 rats, n=750)(p<0.05) of the mast
cells from control sites (Table 1).
EXAMPLE 3
Ultrastructural Analysis of CRH-treated Skin Mast Cells
Ultrastructural observations of mast cells from CRH-injected sites
had obvious sign of activation as evidenced by loss of the electron
dense content of mast cell secretory granules. The effect of CRH on
both vasodilation and mast cell activation was dose-dependent from
10.sup.-4 M-10.sup.-8 M (Table 1).
EXAMPLE 4
Effect of CRH Receptor Antagonist
The peptide CRH-receptor antagonist [D-phe.sup.12, Nle.sup.21,38,
Ala.sup.32 ]rCRH(12-41) did not block the effect of CRH, but at
10.sup.-4 M mimicked (25% activation) the effect of CRH although it
was less potent both as measured by plasma extravasation and mast
cell degranulation (Table 1). This antagonist (10.sup.-4 M) could
not inhibit dye extravasation even when CRH was used at 10.sup.-6 M
(Table 3).
The inactive free acid analogue of CRH, r/h CRH--OH, however,
produced no significant effect on mast cell degranulation (Table 1)
suggesting that these pro-inflammatory actions of CRH are mediated
by specific membrane receptors recognizing the amidated C terminal
part of natural CRH.
EXAMPLE 5
Effect of Somatostatin and CRH on Extravasation
Somatotropin is a general anti-secretory polypeptide hormone.
Pretreatment of the injection site with somatostatin (10.sup.-4 M)
before injecting CRH (10.sup.-4 M) resulted in more dye
extravasation than CRH alone (Table 2). In fact, somatostatin alone
caused significant fluid extravasation which was as strong as that
seen with CRH and was still apparent at 10.sup.-12 M (Table 4).
EXAMPLE 6
Effect of Histamine-1 Receptor Antagonist on CRH Extravasation
While pretreatment with the histamine-1 receptor antagonist
diphenhydramine (10.sup.-4 M) suppressed plasma extravasation at
lower concentrations (<10.sup.-8 M) of CRH, this effect was only
partially inhibited at higher concentrations of (>10.sup.-6 M)
CRH (Table 2). Pretreatment with the non-sedating piperidine H-1
receptor antagonist terfenadine (10.sup.-4 M) reduced the CRH
effect slightly (Table 2), while pretreatment with the piperazine
histamine-1 receptor antagonist hydroxyzine (10.sup.-4 M) and the
tricyclic antidepressant doxepin (10.sup.-4 M) blocked the CRH
effect entirely (Table 2). These results suggest that mast cell
release of histamine is a primary mechanism whereby CRH induces
plasma extravasation.
EXAMPLE 7
Effect of Sensory Nerve Termini Destruction on CRH Effects.
In animals that had been treated neonatally with capsaicin to
destroy neuropeptide-containing sensory nerve termini, mast cell
activation by CRH (10.sup.-4 M) was unaffected, indicating that its
action was not due to neuropeptides such as Substance P.
EXAMPLE 8
Comparison of Mast Cells From Different Tissues
In order to ascertain whether CRH affects mast cells directly, the
effects of CRH on purified rat peritoneal and pleural mast cells
were compared to the effects of C48/80 and Substance P. CRH was
largely ineffective on peritoneal mast cells, but induced histamine
release from pleural mast cells in a dose-dependent manner which,
like Substance P, required the absence of extracellular calcium
ions (Table 5). Comparison of
EXAMPLE 9
Effect of Psychological Stress on Skin Mast Cells
Acute psychological stress due to immobilization of the test
animals also activated skin mast cells, an effect blocked by
pretreatment with anti-serum to CRH.
EXAMPLE 10
Identification of CRH-Positive Nerve Endings in Skin
Occasional CRH-positive nerve endings were identified in human skin
using a polyclonal anti-CRH serum. Human skin is known to express
mRNA for CRH and CRH receptor (Slominski, 1995 above).
Additionally, human mast cells grown in culture were shown with
polymerase chain reaction to contain message for the receptor
(Webster et al., Endocrin. Soc. Abs. 77:669 (1995)).
Summary
These results clearly demonstrate that CRH induces rat skin mast
cell activation which results in fluid extravasation. They further
show that this activation is due to a direct effect of CRH on mast
cells without any involvement of at least sensory neuropeptides. It
is concluded that the skin mast cell is a target of iCRH. Our
hypothesis is that CRH released from peripheral sensory afferent
and/or postganglionic sympathetic nerves acts on local mast cells
and other resident cells to elicit pro-inflammatory responses. The
inflammatory mediators released could further stimulate mast cells,
recruit circulating immune cells to the inflammatory site, activate
local immune accessory cells and act on the nerve endings to
release more inflammatory peptides. This could be of particular
importance in inflammatory states triggered by acute stress, such
as uticaria or eczema, in which activation of the sympathetic
system and local secretion of CRH could lead to mast cell
degranulation and initiation of a new episode or exacerbation of
chronic disease.
The present results may help explain the pathophysiology of certain
neuroinflammatory disorders, such as eczema, pruritus and
urticaria, that are exacerbated by stress. Novel non-peptide
CRH-receptor antagonists or other molecules that could interfere
with CRH-induced skin mast cell degranulation may be useful for the
treatment of such conditions
TABLE 1 ______________________________________ Skin Mast Cell
Activation by CRH Mast Cell Degranulation* Dye (% total)
Extravasation % Conditions (color Total Deg. total (n = 3 rats)
intensity) MC MC deg. ______________________________________ Normal
saline - 750 206 28 C48/80 (0.5 .mu.g/ml) +++ 841 375 45
CRH-.alpha. (10.sup.-4 M) + 112 28 25 CRH-OH (10.sup.-4 M) - 144 13
9 CRH (10.sup.-4 M) ++++ 902 426 49.sup..sctn. CRH (10.sup.-5 M)
+++ 915 327 37 CRH (10.sup.-6 M) ++ 810 331 42 CRH (10.sup.-7 M) ++
1008 329 37 CRH (10.sup.-8 M) ++ 790 254 32 CRH (10.sup.-9 M) + 725
202 28 CRH (10.sup.-10 M) .+-. 880 239 26
______________________________________ *3 sections were examined
from each of four blocks from each rat. .sup..sctn. p < 0.05
compared to normal saline or CRHOH, an inactive form of CRH.
TABLE 2 ______________________________________ Effect of Drugs on
Skin Mast Cell Activation by CRH* Dye Extravasation Conditions
(color (n = 3) intensity) ______________________________________
Normal saline - C48/80 (0.5 .mu.g/ml) +++ CRH +++ TNF receptor
blocker + CRH +++ Diphenhydramine (10.sup.-4 M) + CRH + Doxepin
(10.sup.-4 M) + CRH - Hydroxyzine (10.sup.-4 M) + CRH - Terfenadine
(10.sup.-4 M) + CRH ++ Somatostatin (10.sup.-4 M) + CRH ++++
______________________________________ *used at 10.sup.-4 M
.function. pretreatment with drug for 5 mins.
TABLE 3 ______________________________________ Effect of CRH.alpha.
on Skin Mast Cells* CRH .alpha. concen- Dye tration Extravasation
______________________________________ 10.sup.-4 M +++ 10.sup.-5 M
++ 10.sup.-6 M ++ 10.sup.-7 M .+-. 10.sup.-8 M - 10.sup.-9 M -
10.sup.-10 M - ______________________________________ *n = 3
rats
TABLE 4 ______________________________________ Effect of
Somatostatin on Skin Mast Cells* Somatostatin, Dye concentration
Extravasation ______________________________________ 10.sup.-4 M
+++ 10.sup.-5 M ++ 10.sup.-6 M ++ 10.sup.-7 M ++ 10.sup.-8 M +
10.sup.-9 M .+-. 10.sup.-10 M .+-.
______________________________________ *n = 3 rats
TABLE 5 ______________________________________ Effect of CRH on
Purified Mast Cells Histamine Release (% total) Pleural Peritoneal
Conditions + Calcium - Calcium + Calcium Calcium
______________________________________ Locke's 2.205 4.478 2.0 4.9
Solution C48/80 (10.sup.-4 M) 86.045 67.12 NT NT C48/80 (10.sup.-5
M) 75.552 61.059 NT NT C48/80 (10.sup.-6 M) 47.537 45.203 64.2 NT
SP (10.sup.-4 M) 49.346 54.591 NT NT SP (10.sup.-5 M) 22.919 28.871
NT 39.5 SP (10.sup.-6 M) 2.088 8.538 NT NT CRH (10.sup.-4 M) 22.06
34.244 6.3 25.5 CRH (10.sup.-5 M) 2.102 8.579 1.9 6.9 CRH
(10.sup.-6 M) 1.411 4.253 2.0 4.9
______________________________________ NT = not tested
* * * * *